Qualitative bacteria culture medium identification



United States Patent "Ice Patented jiiffilj aqueous 50.0 percent glucose solution is added per tube 3,399,115 (2 drops from a ml. pipette). The butt is stabbed and g aggg gg gggg MEDIUM the slant streaked in the usual procedure that is used to inoculate other tubed media having a slant and deep butt.

Walter M. Sellers, Jr., 525 S. Pinto St, Although sev ral of the components of the medium San Antonio, Tex. 78207 N0 Drawing Filed Feb 14, 1966, Sen No. 528,026 could be varied both quantitatively and qualitatively and 3 C]aims 195 103 5 still allow results similar to those outllned above under the title, How Accomplished, the composition given ap- The invention described herein may be manufactured pa ently rodu es the best balanced medium. This opinion and used y for the United States Government for V- 10 Was arrived at after a considerable amount of testing. ernmental purposes without the payment to me of any Not only is no single medium available, as previously royalty thereon. stated, for the identification of all of these nonfermenta- This iIlVfiIltiOIl relates to a Single Culture medium for tive bacteria, but neither is a single medium available for assisting in accomplishing the differentiation and the identhe identification of any one of them except for Ps. aerutification of stipulated gram negative nonfermentative 5 girtosa. Two commercial media are available (since Janubacteria of hospital importance, as well as certain other ary 1962) as specific aids in identifying Ps. aeruginosa. pathogenic micro-organisms. Each of these two media depends upon a single charac- The object of the present invention is to provide a te-ristic (pyocyanine or fiuorescein production) for the single culture medium which, with 24 hours, will substanidentification. If a particular strain fails on one of these tially distinguish from one another and identify the folmedia to produce the characteristic feature (and some do lowing species of bacteria of hospital importance: Pseudofor each medium), it will not be identified by that medimonas aeruginosa, Herellea vaginicola, Bacterium anitraum. The described medium has several parameters so M m p y p Alcaligenes faecalis. This that if one characteristic should not occur, the others are object has not been known to have been accomplished ilabl t till ake th identification. previously with any other culture medium. The process 25 Advantage has been taken of the following facts in depresumes the primary isolation of cultures of optimum veloping this medium (most of these are from my repurity. rearch; some are available in the literature).

The intended identification is accomplished with refer- Some strains of Pseudomonas aeruginosa can reduce nience to the following chart: trate but not nitrite to gaseous nitrogen of the symbol N HOW ACCOMPLISHED N2 gas Bluish- Color at production Greenish white flu- Color junction Color (blue or yellow iluorescence of slant of butt of butt green butt crescent at junction and slant and agar slant of butt cracked) and slant Uninoculated medium Green Green Green"... Negative Negative... Negative. Pseudomonas aer'uginos Olive. Blue Bl Do. Herellea vagz'm'cola H Blue Positive Bacterium am'tratum typical strains o do Negative Bacterium am'tratum strains unable to attack L- Yelloi. do do do ..do. Do lllitit a go lzlnorpha Blue Blue .d0 (lo do Do. Alcall'ge7zesfaecalis d0 do Blue Positive do Do.

MEDIUM COMPOsITION PER LITER others can reduce nitrite but not nitrate to N the ma- Water m1 1000 5 jority can reduce both to N Very few species of bacteria Sodium chloride "grams" 2.0 submitted to hospital laboratories are capable of pro- Sodium nitrate ..do 1.0 ducing N (probably only Pseudomonas aeruginosa and Sodium nitrite do 0.35 Alcaligenes faecalis). Although an obligate aerobe under D-mannitol do 2.0 strict conditions, Ps. aeruginosa can grow anaerobical y L-arginine do 10 in the presence of nitrate and produce sufficient ammonia Yeast extract do 1.0 in the described medium to change a pH indicator (alka- Magnesium sulfate 7H O do 1.5 line butt). Pseudomo'nas aeruginosa is very proteolytic Potassium phosphate 3H O (dibasic) do 1.0 and prefers peptone to glucose as an energy source, hence Bacto peptone do 20.0 the alkaline junction with this medium. Phenol red do 0.008 Mannitol is second only to glucose as a carbohydrate Brom thymol blue do 0.040 energy source for Ps. aeruginosa and has the advantage Bacto agar, pH adjusted to 6.7 d0 15.0 that it stimulates fluorescein production better than other carbohydrates. It is a nonreducing sugar and thus does Gram negative fefmentative bacteria of hospital not interfere with the anaerobic growth of Ps. aeruginosa: portance can be distinguished by their yellow butts with 0 i i di this medium; this also includes those species which cannot Th ll amount f glucose added to the slanted tubes ferment mannitol. before inoculation allows the powerful glucose oxidizers, The ingredients Of the above m dium comp si i P r Bacterium anitratum, Herellea vaginicola, to produce an liter are dissolved in the water, taking care to dissolve the acid (yellow) area in the immediate vicinity, while magnesium sulfate and the potassium phosphate in S a l Pseudomonas aeruginosa, a less powerful glucose oxidizer, amounts of the Water separately before adding them to does not do so because of the high peptone content. The the medium to avoid formation of an insoluble magnesismall amount of glucose transmitted to the butt by the urn phosphate precipitate. The medium composition is stab inoculation is suflicient to cause fermentative orboiled to dissolve the agar and is tubed in amounts sufliganisms to produce acid (yellow) butts even if they do not cient to make 1 /2 inch butts and 3 inch slants before beferment the mannitol (i.e., Proteus vulgaris or P. mirabing autoclaved for 10 minutes at 15 pounds pressure. Imilis).The glucose drops also stimulate Herellea vaginicola mediately before being inoculated, 0.15 ml. of a sterile, to produce afluorescence in the immediate vicinity.

Bibasic potassium phosphate stimulates N production. Three-tenths of one percent was best for N formation but 0.1% was used because it was almost as good for N production; its less buffering effect allowed a faster pH indicator change in the butts (blue color) of the tubes inoculated with Pseudomonas aeruginosa or Alcaligenes faecalz's; it had less tendency to form a precipitate in the presence of magnesium sulfate than did the higher concentration.

Magnesium sulfate stimulates fiuorescein production by Pseudomonas aeruginosa. Fifteen-hundredths of one percent was the Optimum concentration. Less caused a decrease in fluorescence; more tended to precipitate the phosphate in this medium.

Sodium nitrate 0.3% produced the greatest N gas production with most Pseudomonas aeruginosa strains. Even through they produced less gas 0.1% NaNO was used since the 0.3% concentration was toxic for a few strains of this species and interferred with the glucose oxidation by Bacterium anitratum and Herellea vaginicola and with fermentation by Proteus species.

Sodium nitrite 0.035% was optimum; more was toxic and less was ineffective with a number of Pseudomonas acruginosa strains.

Mima polymorpha, Herellea vaginicola, and Bacterium anitratum are obligate acrobes even in the presence of nitrate. Therefore they are incapable of growing in the butt of the tubes and therefore the pH indicator remained unchanged (green) in the butts. Alcaligenes faecalis is a facultative anaerobe and produced an alkaline (blue) butt, as did Pseudomonas aeruginosa (although it is an obligate aerobe) because the nitrate allowed it to grow anaerobically.

As far as is presently known, this is the only culture species produce fiuorescein, several soil species produce gaseous nitrogen, and only Ps. aeruginosa will produce both gaseous nitrogen and fiuorescein.

It is to be understood that similarly functioning modifications may be made in the preferred medium composition that is disclosed herein and that expansions may be made in the bacteria that may be subsequently identified by the composition that is disclosed herein without departing from the spirit and scope of the present invention.

I claim:

1. The qualitative bacteria culture medium identification composition per liter that is substantially:

2. The process of using the culture medium defined in claim 1 for the differentiation and the identification of gram negative nonfermentative bacteria that is accomplished by subjecting the bacteria to the application of the composition defined by claim 1 and applying the observed resultant phenomena to the following chart for the identifiying of the unknown bacteria:

HOW ACCOMPLISHED N 2 gas Bluish- Color at production Greenishwhite flu- Coior junction Color (blue or yellow fiuorescence of slant of butt of butt green butt crescent at junction and slant and agar slant of butt cracked) and slant Uninoculated medium Green Green Green.. Negative N egative Negative. Pseudomonas aerugz'n0sa- 0live Blue Blue Positive Positive. Do. Herellea vaginicola Blue Yellow Green..." Negative.... N egstive.-.. Positive. Bacterium anitmtum typical strains do do do do do.-. Negative. Bacterium am'tratum strains unable to attack L- Yellow -do do do do Do.

argmme. Mimu polymorpha Blu do Do. Alcalz'genes faecalis .41 Do.

medium that is capable of differentiating and of identifying the gram negative, nonfermenting bacteria that is of importance in hospital work.

The process described herein is more reliable for the identification of Pseudomonas aeruginosa than are the two known commercial media that are designated for this purpose. The reasons that the process described herein is believed to be more reliable than commercially available media are in the identification of the bacterial cultures that are designated herein are: (a) the process disclosed herein does not depend upon a single characteristic for the identification; (b) the disclosed process is at least as eflicient in stimulating fiuorescein production as is the commercial medium for the same purpose; (c) more strains of Pseudomonas aeruginosa produce fluorescein than product pyocyanine; (d) of the 150 species of Preudomonas, several References Cited UNITED STATES PATENTS 3,278,393 10/1966 Bahn et a1 l00 A. LOUIS MONACELL, Primary Examiner. L. SCHENKMAN, Assistant Examiner. 

1. THE QUALITATIVE BACTERIA CULTURE MEDIUM IDENTIFICATION COMPOSITION PER LITER THAT IS SUBSTANTIALLY: WATER ML 1000 SODIUM CHLORIDE GRAMS 2.0 SODIUM NITRATE DO 1.0 SODIUM NITRITE DO 0.35 D-MANNITOL DO 2.0 L-ARGININE DO 1.0 YEAST EXTRACT DO 1.0 MAGNESIUM SULFATE 7H2O DO 1.0 POTASSIUM PHOSPHATE 3H2O (DIBASIC) DO 1.0 BACTO PEPTONE DO 20.0 PHENOL RED DO 0.008 BROM THYMOL BLUE DO 0.040 BACTO AGAR PH ADJUSTED TO 6.7 DO 15.0 